Devices and methods for thermally-mediated chemical reactions

ABSTRACT

One aspect of the invention provides container for thermal cycling a plurality of samples in a microfluidic array. The container includes a plurality of walls defining an interior volume and a conductive member for heating the interior volume. Another aspect of the invention provides container for thermal cycling a plurality of samples in a microfluidic array. The container includes a plurality of walls defining an interior volume and a plurality of conductive members for heating an interior volume. Another aspect of the invention provides a container for thermal cycling a plurality of samples in a microfluidic array. The container includes a plurality of walls defining an interior volume and a first conductive member located in the interior volume and adapted to contact a first end of the microfluidic array.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/034,321, filed Mar. 6, 2008. The contents of this patentapplication are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

New apparatus and methods for thermal cycling specimens are providedherein. The methods are applicable to specimens held in a variety oflaboratory vessels and are particularly advantageous when used inconjunction with specimens held in a through-hole array.

BACKGROUND

Various research and diagnostic techniques employ thermally-mediatedchemical reactions such as the polymerase chain reaction (PCR), nucleicacid hybridization, and protein immunoassays and/or thermally-controlledenvironments for cell culture. Such techniques utilize a thermal cycler(also known as a thermocycler, PCR machine, and/or DNA amplifier).Thermal cyclers provide the high temperatures necessary to physicallyseparate the strands of DNA double helix that is used as a template atlower temperatures for DNA synthesis by a DNA polymerase (e.g. Taqpolymerase)) to selectively amplify the target DNA.

Current methods for changing temperature of a liquid contained in amicrotiter plate or sealed tube (e.g. Eppendorf tube or capillary tube)make use of an external, temperature-controlled liquid or solid totransfer heat into or extract heat from the liquid PCR reagents in anenclosed container. The primary limitation of this approach is therequirement for an intervening material between the energy source andthe heated or cooled liquid. Miniaturization and scaling to largernumbers of liquid volumes to be heated and cooled is particularlyproblematic with the current art because of the dual design constraintsof having to seal and heat/cool a large volumes of liquids contained inthe well of a thermoplastic microtiter well plate.

SUMMARY OF THE INVENTION

One aspect of the invention provides container for thermal cycling aplurality of samples in a microfluidic array. The container includes aplurality of walls defining an interior volume and a conductive memberfor heating the interior volume.

This aspect can have a variety of embodiments. The conductive member canbe one of the plurality of walls. The conductive member can be locatedwithin the interior volume. The conductive member can be a metal. Theconductive member can be in communication with one or moreelectrically-conductive contacts located on an exterior surface of thecontainer. The container can include a temperature sensor configured tomeasure a temperature of the microfluidic array. One of the plurality ofwalls can be optically transparent.

Another aspect of the invention provides container for thermal cycling aplurality of samples in a microfluidic array. The container includes aplurality of walls defining an interior volume and a plurality ofconductive members for heating an interior volume.

This aspect can have a variety of embodiments. The plurality ofconductive members can be separated by an insulator. The insulator canbe air or an adhesive. The plurality of conductive members canconstitute one of the plurality of walls. The plurality of conductivemembers can be located within the interior volume. The plurality ofconductive members can be composed of the same material. The pluralityof conductive members can be composed of different materials. Theplurality of conductive members cart have different electricalresistances.

Another aspect of the invention provides a container for thermal cyclinga plurality of samples in a microfluidic array. The container includes aplurality of walls defining an interior volume and a first conductivemember located in the interior volume and adapted to contact a first endof the microfluidic array.

This aspect can have a variety of embodiments. The first conductivemember can be in communication with a first contact located on anexterior surface of the container. The container can include a secondconductive member adapted to contact a second end of the microfluidicarray. The second conductive member can be located within a mechanicalplug configured to substantially seal the container. The secondconductive member can be in communication with a second contact locatedon an exterior surface of the mechanical plug.

Another aspect of the invention provides an apparatus for thermalcycling a plurality of samples in a microfluidic array received in acontainer. The apparatus includes a first and a second electricalcontact and a controller configured to selectively complete theelectrical circuit, thereby beating the microfluidic array. The firstand the second electrical contacts are configured to form an electricalcircuit across the container.

This aspect can have a variety of embodiments. The controller can be aswitch. The apparatus can include a power supply in communication withthe circuit. The apparatus can include a heat sink. The heat sink can bea fluid bath. The fluid bath can be chilled. The heat sink can be aPeltier element. The apparatus can include a temperature sensor formonitoring the thermal cycling of the microfluidic array. Thetemperature sensor can be in communication with the controller.

The apparatus can include an imager. The imager can be charge-coupleddevice. The apparatus can include an illuminator. The illuminator can beone or more light emitting diodes. The illuminator can be a tungsten arclamp.

The apparatus can :include one or more concave mirror configured toilluminate the microfluidic array and an optical fiber bundle configuredto channel light from the tungsten arc lamp to the microfluidic array.The apparatus can include a filter wheel configure to condition lightbefore the light is received by the imager.

Another aspect of the invention provides a method for thermal cycling aplurality of samples in a microfluidic array received in a container.The method includes causing electrical current to flow through thecontainer to heat the samples by Joule heating and terminating the flowof electrical current to allow the samples to cool.

This aspect can have a variety of embodiments. The container can includea plurality of walls. The electrical current can flow through at leastone of the walls. The electrical current can flow through themicrofluidic array within the container. The method can include placingthe container in contact with a heat sink. The step of placing thecontainer in contact with a heat sink can include submerging thecontainer in a fluid bath. The step of placing the container in contactwith a heat sink can include placing the container against a Peltierelement. The method can include imaging the plurality of samples.

Another aspect of the invention provides a method for thermal cycling aplurality of samples in a microfluidic array received in a container.The method includes exposing the microfluidic array to radiation to beatthe plurality of samples and terminating the radiation exposure to allowthe samples to cool.

This aspect can have a variety of embodiments. The method can includerepeating the exposing and terminating steps a plurality of times. Theradiation can be microwave radiation or infrared radiation.

Another aspect of the invention provides a through-hole array including:a platen having a first end region a second end region, a plurality ofstrips spanning from the first end region to the second end region, anda plurality of through-holes located on one or more of the plurality ofstrips.

This aspect can have a variety of embodiments. The strips can besubstantially parallel. The through-hole array can include one or moreslots. Each slot can separate two of the plurality of strips. The platencan be formed from a conductive material. The conductive material can beselected from the group consisting of: copper, gold, silver, nickel,iron, titanium, steel, and stainless steel. The plurality ofthrough-holes can be located on one of the plurality of strips arearranged in a single column. The through-holes can have a hydrophilicinterior. The through-hole array can include two outer layers ofhydrophobic material coupled to a top and a bottom surface of theplurality of the strips. Each of the plurality of through-holes can havea volume less than 100 nanoliters.

Another aspect of the invention provides a container for thermal cyclinga plurality of samples in a microfluidic array having a plurality ofthrough-holes arranged on a plurality of strips. The container includes:a plurality of walls defining an interior volume and a plurality offingers configured to contact the strips when the microfluidic array isinserted in the container.

This aspect can have a variety of embodiments. The container can includea pair of electrically-conductive contacts located on an exteriorsurface of the container. The contacts can be in communication with thefingers. The fingers can be configured to contact the microfluidic arrayat a first and a second end of each of the plurality of strips. At leastone of the plurality of walls can be optically transparent. Theplurality of fingers can be comprised of a metal.

Another aspect of the invention provides a method for thermal cycling aplurality of samples. The method includes providing a through-hole arrayincluding a platen having a first end region and a second end region, aplurality of strips spanning from the first end region to the second endregion, and a plurality of through-holes located on one or more of theplurality of strips; loading the plurality of samples into the pluralityof through-holes; placing the though-hole array in a container; applyinga flow of electrical current across the plurality of strips; andterminating the flow of electrical current to allow the samples to cool.The container includes a plurality of fingers configured to contact thestrips.

This aspect can have a variety of embodiments. The method can includeplacing the container in contact with a heat sink. The heat sink can bea fluid bath. The fluid bath can be chilled. The heat sink can be aPeltier element. The method can include imaging the through-hole array.

Another aspect of the invention provides a container for thermal cyclinga plurality of samples in a microfluidic array. The container includes;a plurality of walls defining an interior volume, a first port locatedan a first end of the container, and a second port located on a secondend of the container. The first port and the second ports are configuredto provide fluid communication with the interior volume.

This aspect can have a variety of embodiments. The second end can be asubstantially opposite end of the container with respect to the firstend. The first port and the second port can each include a gasketconfigured to prevent fluid flow when the container is not coupled witha thermal cycler. The container can include a plurality of veins locatedwithin the interior volume to promote uniform fluid flow.

Another aspect of the invention provides an apparatus for thermalcycling a plurality of samples in microfluidic array. The apparatusincludes a hot liquid source; a cold liquid source; a pump in fluidcommunication with the hot liquid source and the cold liquid source; afluidic circuit coupled to the hot liquid source, the cold liquidsource, and the pump; and an interface adapted to couple the fluidiccircuit to a container housing the microfluidic array. The containerincludes a first port and a second port.

This aspect can have a variety of embodiments. The hot liquid source canbe a tank. The hot liquid source can be a heater. The cold liquid sourcecan be a tank. The cold liquid source can be a chiller.

The apparatus of claim 1 can include an imager. The imager can be acharge-coupled device. The apparatus can include an illuminator. Theilluminator can include one or more light emitting diodes (LEDs). Theilluminator can be a tungsten arc lamp. The apparatus can include: oneor more concave mirror configured to illuminate the microfluidic arrayand an optical fiber bundle configured to channel light from thetungsten arc lamp to the microfluidic array. The apparatus can include:a filter wheel configure to condition light before the light is receivedby the imager.

Another aspect of the invention provides a method for thermal cycling aplurality of samples. The method includes: loading the plurality ofsamples in a microfluidic array; placing the microfluidic array in acontainer, the container comprising a plurality of walls defining aninterior volume, a first port located on a first end of the container,and a second port located on a second end of the container, wherein thefirst port and the second ports are configured to provide fluidcommunication with the interior volume; coupling the first port and thesecond port to a fluidic circuit including a pump, a hot fluid source,and cold fluid source; and alternatively pumping a hot fluid and a coldfluid through the container.

This aspect can have a variety of embodiments. The method can includeapplying a layer of an immiscible liquid over the microfluidic array.The method can include sealing the container.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature and desired objects of thepresent invention, reference is made to the following detaileddescription taken in conjunction with the accompanying drawing figureswherein like reference characters denote corresponding parts throughoutthe several views and wherein:

FIG. 1 depicts an exemplary embodiment of a through-hole array for usewith various embodiments of the invention,

FIGS. 2A-2C depict a case for holding a through-hole army according toone embodiment of the invention.

FIG. 3 depicts a thermal cycler for the rapid thermal cycling of samplesheld in a through-hole array within a case according to one embodimentof the invention,

FIG. 4A depicts an embodiment of a through-hole array in whichthrough-holes are arranged on strips according to one embodiment of theinvention. FIG. 4B depicts the application of a plurality of conductivefingers to contact the end of strips on the through-hole array to directelectrical current through the strips according to one embodiment of theinvention.

FIG. 5 depicts a system that provides optical access to a through-holearray while the array is heated and cooled according to one embodimentof the invention.

FIG. 6 depicts a system that provides optical access to a through-holearray from a single light source while the array is heated and cooledaccording to one embodiment of the invention.

FIGS. 7A and 7B depict fast scanning systems according variousembodiments of the invention.

FIGS. 8 and 9 depict thermal control devices according to variousembodiments of the invention,

FIGS. 10A and 10B depict schemes from controlling the flow of currentthrough a platen according to one embodiment of the invention.

FIGS. 11A and 11B illustrate a one-dimensional thermal model forestimating the temperature decrease of a platen during Joule heating bya current pulse according to one embodiment of the invention.

FIG. 12 depicts a case containing three conductive strips according toone embodiment of the invention.

FIG. 13 depicts a system for thermal cycling a through-hole arrayaccording to one embodiment of the invention.

FIG. 14 depicts a method for thermal cycling a plurality of samplesaccording to one embodiment of the invention.

DEFINITIONS

The instant invention is most clearly understood with reference to thefollowing definitions:

As used in the specification and claims, the singular form “a”, “an” at“the” include plural references unless the context clearly dictatesotherwise.

The term “biocompatible” denotes a natural or artificial substrate thatsupports cellular adhesion or proliferation without eliciting a toxic orother undesirable effect in a cell in contact with the substrate.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

New apparatus and methods for thermal cycling, specimens are providedherein. The methods are applicable to specimen held in a variety oflaboratory vessels and are particularly advantageous when used inconjunction with specimens held in a through-hole array,

Through-Hole Arrays

Through-hole arrays generally consist of a platen having a plurality ofthrough-holes extending from a first surface of the platen to a secondsurface of the platen. The platen can be fabricated from a variety ofmaterials including metals (e.g. copper, gold, silver, nickel, iron,titanium., and alloys thereof such as steels and stainless steels),plastics, conductive silicon, glass, other rigid materials, semi-rigidmaterials, flexible materials, and the like.

FIG. 1 shows a cutaway view of a exemplary microfluidic through-holearray of through-holes. The sample array 100 includes a thin plate ofmaterial 102 having a pair of opposed surfaces 104, 106 and a thickness.A large number of through-holes 108 (e.g. 384 or 3,072 through-holes)penetrate through the thickness from one of the surfaces 104 to theother opposing surface: 106 (not shown). Through-holes 108 can, in someembodiments, be arranged in groups (e.g. 12×4 groups of 8×8through-holes 108) for ease of loading, reference, and interfacing withstandard 96- or 384-well microtiter plates.

The sample array 100 can, in some embodiments, have a thickness T from0.1 mm to more than 10 mm, for example, around 0.3 to 1.52 ram or 0.5mm. Typical volumes of the through-holes 12 can range from 0.1 picoliterto 1 microliter, with common volumes in the range of 0.2-100 nanoliters,for example, about 35 nanoliters. Capillary action or surface tension ofthe liquid samples can be used to load the sample through-holes 108. Fortypical plate dimensions, capillary forces exceed gravitational andinertial forces on the liquid retained in each hole. Plates loaded withsample solutions can readily handled and even centrifuged at moderatespeeds without displacing samples.

The use of through-holes 108, as compared to closed-end well structures,reduces the problem of trapped air inherent in other microplatestructures. The use of through-holes together with hydrophobic andhydrophilic patterning enables self-metered loading of the samplethrough-holes 108. The self-loading functionality helps in themanufacture of arrays with pre-loaded reagents, and also in that thearrays will fill themselves when contacted with an aqueous samplematerial,

Suitable through-hole arrays are available under the OPENARRAY®trademark from BioTrove, Inc. of Woburn, Mass. and are described in U.S.Pat. Nos. 6,306,578; 6,387,331; 6,436,632; 6,716,629; 6,743,633;6,893,877; 7,332,271; and U.S. Patent Application Publication Nos.2001/0055765; 2002/0151040; 2002/0192716; 2003/0124716; 2003/0180804;2004/0037748; 2004/0171166; 2004/0191924; 2004/0208792; 2005/0059074:2005/0079105; 2005/0148066; 2005/0230213; 2006/0105453; 2006/0183171;2007/0003448; and 2008/0108112.

Coated Through-Hole Arrays

To enhance the capillary action of the through-holes 108, the targetarea of the receptacle, interior walls 110, can have a hydrophilicsurface that attracts a liquid sample. It is often desirable that thesurfaces be biocompatible and not irreversibly bind biomolecules such asproteins and nucleic acids, although binding may be useful for someprocesses such as purification and/or archiving of samples.Alternatively, the sample through-holes 108 can contain a poroushydrophilic material that attracts a liquid sample. To preventcross-contamination (crosstalk) between the through-holes, the exteriorplanar surfaces 112 of plate 100 and a layer of material 114 around theopenings of sample through-holes 108 can be hydrophobic or can be coatedwith a hydrophobic material. In one embodiment, the interior walls 110are made hydrophilic by covalently linking polyethylene glycol (PEG) orother similar hydrophilic and biocompatible molecules to the surface andthe exterior 114 is made hydrophobic by covalent bonding offluoroalkylsilane or similar hydrophobic molecules to the surface. Thus,each through-hole 108 can have an interior hydrophilic, biocompatibleregion 110 bounded at either end by a hydrophobic region 114.

Exemplary methods for coating through-hole arrays are briefly describedbelow and are described in greater detail in U.S. Patent ApplicationPublication No. 2006/0105453.

In one embodiment, one or more plates are first cleaned in a solution ofabout 10% RBS®-35 detergent, available from Thermo Fisher Scientific Incof Rockford, Ill., at about 50° C. for about two hours, C8 vinylsilane(7-octenyltrimethoxysilane) is then applied through vapor deposition forabout 2.5 hours at about 150° C to form a reactive vinyl monolayer.

In order to introduce oxidizing solution into the through-holes toremove the hydrophobic vinyl monolayer, a “forced loading” technique canbe used. The plates are first dipped in a lower energy (surface tension)liquid such as ethanol, which is retained in the through-holes. Theplates are then immersed in water, which replaces the ethanol in thethrough-holes. The plates are then placed in a loading chambercontaining an oxidizing solution containing about 360 mL of about 5 mMKMnO₄ and about 40 mL of about 19.5 mM NaIO₄ floating on an immisciblefluid such as FLUORINERT® FC-3283. The fluid level is raised to fill thethrough-holes with the KMnO₄/NaIO₄ solution. The plates are thenincubated for about two hours. The plates are then placed in a chambercontaining a solution containing 400 ml of 1.5 mg/mL EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride) and 5 mg/mLpolyethylene glycol (PEG) floating on an immiscible fluid such asFLUORINERT® FC-3283. The solution level is again raised to fill thethrough-holes with the solution and the plates are again incubated forabout two hours in the solution. The plates are then dried overnight atabout 100° C under vacuum with any EDC and PEG present in the solution.

The plates are then reloaded with a solution containing about 50 mg/mLof a high-weight PEG (e.g. PEG 8000) floating on an immiscible fluidsuch as FLUORINERT® FC-3283 so that the through-holes are filled withthe solution. The fluid level is lowered to remove the PEG solution fromthe through-holes before removing the plates from the chamber. Theplates are then dried for about three hours at about: 100° C undervacuum.

A hydrophobic coating is re-applied by placing the plates in a vapordeposition chamber for about two hours at about 150° C and exposed toperfluorotriethoxysilane and/or vinylsilane. The plates are then curedin gaseous ammonia for about 30 minutes. The resulting hydrophobicsurfaces can be characterized by contact angles about 90°, for examplegreater than about 100°, about 110°, about 120°, about 130°, about 140°,about 150°, about 160°, about 170°, or about 180°. The plates are thenrinsed to remove excess physisorbed PEG from the through-holes alongwith the perflurosilane film deposited on the top excess PEG.

In some embodiments, the plates are cleaned in an RBS®-35 solution asdescribed and placed in a microwave-generated plasma with a trace amountof water to remove any organic debris on the plate surface and tofunctionalize the surface with hydroxyl groups. The plates are thenfurther processed as described.

Exemplary Uses of Through-Hole Arrays

In various embodiments, through-hole arrays can be used as follows.Reagents for implementing different biochemical or biological analysesof one or more biological samples are loaded into the through-holes. Forexample, in the case of PCR analyses, each through-hole can contain adifferent primer pair or different primer-probe set. Biological samplesmixed with PCR reagents (e.g. MASTERMIX® reagents available from AppliedBiosystems of Foster City, Calif.) are then loaded into one or morethrough-holes.

The through-hole array is then inserted into a case containing animmiscible, optically transparent liquid that acts as an evaporationbarrier. Suitable immiscible liquids include FLUORINERT® coolants (e.g.FLUORINERT® FC-77, having a chemical formula of C₈F₁₉), silicon oil, ormineral oil, FLUORINERT® coolants are available from the MinnesotaMining and Manufacturing Company of St. Paul, Minn. Other desirablephysicochemical properties of the immiscible liquid are a moderatethermal conductivity and a low electrical conductivity.

Through-Hole Array Cases

FIGS. 2A, 2B, and 2C depict a case 200 for holding a through-hole arrayaccording to one embodiment of the invention. FIG. 2A provides a topview of the case 200, while FIGS. 2B and 2C provide cross-sectionalviews of the case 200. Case 200 is designed to hold a through hole array202, which can contain a plurality of groups 204 a-204 h ofthrough-holes (not depicted).

Case 200 can be formed from a variety of materials capable of holdingthrough-hole array 202 and immiscible liquid (not depicted). In oneembodiment, the case 200 is fabricated from two platens 206 a, 206 bconnected by one or more gaskets 208 a-208 d. The platens can include avariety of materials such as glass, plastics, metals, and the like. Insome embodiments, at least one of platens 206 a, 206 b are opticallytransparent to allow for monitoring of reactions in the through-holearray during thermal cycling or during isothermal amplificationreactions. Additionally or alternatively, one of the platens 206 a, 206b can be optically opaque and/or non-reflective to minimize unwantedreflections when imaging.

In various embodiments where the rapid heating and/or cooling of thethrough-hole array 202 is desired, thinner platens 206 a, 206 b can beused. Thermal conduction through glass scales in proportion tothickness. Accordingly, the use of microscope slide covers, which areabout 170 micrometers thick, facilitates rapid heat transfer. Coolingrates of at least 10° C./second can be achieved utilizing a caseconstructed in this manner and the systems and methods described herein.

In another embodiment, one or more of the platens 206 a, 206 b can becomposed of a material with a high thermal conductivity. For example,one or more of the platens 206 a, 206 b can be composed of metals suchas gold, silver, copper, iron, brass, aluminum, and allows thereof. Tofurther increase conduction, one or more of the platens 206 a, 206 b caninclude one or more vanes or protuberances to increase the surface areain contact with a heat sink.

The gaskets 208 a-208 d can be formed from a variety of materials suchas plastics, rubbers, resins, metals, and the like. In one embodiment,one or more of the gaskets 208 a-208 d are a liquid crystal polymer(LCP), for example, a liquid crystal polymer containing 40% glass fiber.Suitable LCPs are available under product number RIP 3407-3 from RTPCompany of Winona, Minn. The gaskets 208 a-208 d and platens 206 a, 206b can be bonded with a variety of adhesives selected for the particularmaterials of the gaskets 208 a-208 d and platens 206 a-206 b. In someembodiments, gaskets 208 a-208 d and platens 206 a 206 b are be bondedwith transfer adhesive- tape (e.g., 3M® Adhesive Transfer Tape 468MP,available from the Minnesota Mining and Manufacturing Company of St.Paul, Minn. In some embodiments, the gaskets 208 a-208 d and platens 206a 206 b are plasma irradiated within 24 hours prior to assembly toimprove bonding strength.

Although depicted as four distinct gasket components 208 a-208 d, one ormore gasket components 208 a-208 d can be combined into a single gasketcomponent. For example, gasket components 208 a, 208 b, 208 c can be asingle U-shaped gasket, while gasket component 208 d is inserted afterthe through-hole array 202 is inserted into the case 200. One or moregasket components can be composed entirely from an adhesive (e.g. aUV-curable adhesive such as DYMAX® OP-29V, available from DYMAXCorporation of Torrington, Conn.) applied after the through-hole array202 is inserted into the case 200. In another embodiment, gasketcomponents 208 a-208 d form a single gasket and platen 206 a or 206 b isbonded to the gasket after the through-hole array 202 is inserted.

As depicted in FIGS. 2A-2C gasket components 208 a-208 d can optionallyhave a U-shaped groove to hold the through-hole array 202 away fromplatens 206 a 206 b. A variety of other suitable case designs andfeatures are described in U.S. Patent Application Publication Nos.2004/0208192 and 2006/0094108.

In one embodiment of the invention, Joule heating is used to heat thesamples held in through-hole array 202 during the thermal cyclingprocess or to bold the plate at a constant temperature for isothermalamplification. To facilitate electrical flow through the through-holearray 202 while protecting the samples held therein from evaporation bycase 200, one or more gasket components (e.g. 208 a and 208 b, or 208 cand 2080) can include electrically conductive materials. In someembodiments, the gasket components 208 a-208 d include distinctconductive portions (e.g. wires extending from an interior portion ofthe gasket component 208 a-208 d in contact with the through-hole array202 to an exterior portion of the gasket in contact with an electricalsource). In other embodiments, the gasket is composed of an electricallyconductive material such as conductive resin (e.g. a resin impregnatedwith a conductive material such as copper, gold, silver, nickel,stainless steel, nickel-coated graphite, carbon black, carbon powder,carbon fibers, and the like). Conductive resins are available from RTPCompany of Winona, Minn. and Cool Polymers of Warwick, R.I. In anotherembodiment, Joule heating of the platen is accomplished by inductionheating by radio or microwave frequency radiation.

One or more gasket components 208 a-208 d can be hermetically sealed toplatens 206 a 206 b so that 310 liquid leaks from the case. However, notall gasket components 208 a-208 d need to form a leak-tight hermeticseal. For example relaxing the requirement that “top” component of thegasket (e.g. 208 d) be leak-tight during temperature cycling simplifiesthe case design, manufacture use.

Thermal Cyclers

Referring to FIG. 3, a thermal cycler is provided for the rapid thermalcycling of samples held in a through-bole array 202 within a case 204.The case 200 is connected to electric leads 304 a, 304 b. When switch306 is closed, electricity flows from power source 308 through case 204and/or through-hole array 202. The electrical resistance of case 204and/or through-hole array 202 generates heat, which is conductivitytransferred to the samples contained therein.

The through-hole array 202 can be cooled during the annealing phase by avariety of means. For example, the case 204 can be in contact with theatmosphere.

In order to speed cooling, an active cooling means such as a fan can beemployed. In the illustrated embodiment of PIG. 3, the case 200 isplaced within a vessel 302 containing liquid 304. The liquid 304 isideally an optically transparent liquid with a high heat capacity suchas water or refrigerant such as FLUORINERT® coolant (e.g. FLUORINERT®PC-70 or FC-77) or a water/ethanol mixture.

Vessel 302 can be a closed vessel or can be open to the atmosphere.Vessel 302 can be wholly or partially formed from a opticallytransparent material such as glass. In some embodiments, portions of thevessel 302 can he optically opaque and/or non-reflective to reduceambient reflections that could interfere with imaging of the samples.

The refrigeration liquid 304 is cooled to a low and substantiallyconstant temperature by a refrigerator unit 310 to a temperature abovethe liquid freezing point, making the temperature difference between theplaten 202 and liquid heat sink 304 large. This, in turn, facilitatesrapid heat flow from the heated platen 202 into the cooled liquid 304.

The temperature of the through-hole array 202 can, in some embodiments,be monitored to verify that the samples are sufficiently heat& Forexample, a temperature sensor, such a thermometer, a bi-metal mechanicalthermometer, a thermocouple, a liquid crystal thermometer, and the likecan contact the through-hole array, the case, and/or the liquid 304.Alternatively, an infrared thermometer can measure the temperature ofthe through-hole array and/or the samples. In another embodiment, athermistor can be used to measure the temperature based on changes inthe resistance of the through-hole array 202 and/or case 204 as thetemperature changes.

One method for heating and cooling the array to implement the polymerasechain reaction (PCR) is described below. PCR is described in greaterdetail in a variety of publications such as Shadi Mahjoob. Rapidmicrofluidic thermal cycler for polymerase chain reaction nucleic acidamplification, 51 Int'l J. of Heat and Mass Transfer 2109-22 (2008).

Starting at a basal temperature, switch 306 is closed and an electricalcurrent (e.g. about 100 Amps (A), about 200 A, about 300 A, about 400 A,about 500 A, about 600 A, and the like) is injected into thethrough-hole array 202, heating the array 202 and the liquid retained inthe through-holes. After the through-hole array reaches the desiredtemperature (e.g. 98,26° C the melting temperature for double-strandedDNA), the switch opens to break the current flow. In some embodiments ofthe invention, heat conduction from the array rapidly drops the liquidtemperature to a lower value (e.g. 55° C the annealing temperature forDNA). When the array temperature has dropped to a prescribed level, theswitch 306 is closed, current is re-injected the array 202 and resistiveheating increases the array temperature to a higher set-point level(e.g. 98.26° C the melting temperature for double-stranded DNA). Variousembodiments of the invention can heat the array to a melting temperaturefor double-stranded DNA in less than one second (e.g. about 200 ms,about 250 ms, about 300 ms, about 350 ms, about 400 ms, about 450 ms,about 500 ms, about 550 ms, about 600 ms, about 650 ms, about 700 ms,about 750 ms, about 800 ms, about 850 ms, about 900 ms, about 950 ms,and the like). This process can be automated and performed cyclically toincrease/decrease temperature of the array as required forimplementation of the PCR assay or to hold the array at a constanttemperature, above or below ambient temperature, as prescribed forisothermal reactions or cell culture.

Temperature differences resulting from non-uniform current injection anthermal conductivities of the through-hole array 202 and/or the case 200can be compensated for by injecting current at different points on thethrough-bole array 202 and/or the case 200. For example, instead ofinjecting, current at one end and exiting the opposing end, lengthwisealong the through-hole array 202 and/or the case 200, current can beinjected along the long side of the through-hole array 202 and/or thecase 200 and exit from the opposing edge.

Alternatively, the through-hole spatial distribution, size, geometry andspacing can be modified to achieve uniform heating. FIG. 4A depicts oneexemplary embodiment of such a through-hole array 400. A plurality ofthrough-holes 402 a, 402 b and slots 404 a-404 eare formed such that thethrough-holes 402 a, 4026 are arranged on strips 406 a-406 d. Asdepicted in FIG. 4B, to heat the through-hole array, a plurality ofconductive fingers 408 a-408h contact the end of strips 406 a-406 d todirect most of the electrical current (represented by arrows) throughstrips 406 a-406 d.

Isothermal Amplification

In another embodiment of the invention, the temperature of the plate isheld at a substantially constant temperature (e.g. about 42° C. for theTranscription Mediated Amplification (TMA) assay) by a series of currentpulses modulated by feedback from a thermal sensing element (e.g., athermal sensing element in contact with the platen).

Through-Hole Array Imaging System

FIG. 5A depicts an embodiment of a system 500 a that provides opticalaccess to a through-hole array 202 while the through-hole army 202 isheated and cooled. System 500 can implement protocols such as thereal-time PCR process for quantitative measurement of gene expression,isothermal amplification for quantitative measurement of geneexpression, and/or recordation of activities of cells loaded into eachchannel of the array.

In system 500, a pair of light emitting diodes (LEDs) 502 a, 502 bobliquely illuminate the way 202 in a transillumination configurationand the light: either passed through the array channels 504 or emittedfrom each through-hole 504 of the array (e.g. fluorescence) is recordedby an electronic camera 506. A filter wheel 508 containing neutraldensity or spectrally selective filters is optionally included tointensify or spectrally condition the light before it is recorded by thecamera 506 (e.g. a charge-coupled device, referred to herein as a“CCD”).

The LED light sources 502 a, 502 b are controlled by an electroniccontroller 510 via wires 512 a, 512 b and synchronized to thetemperature modulation of the through-hole array 202 to effect a givenapplication. Electronic controller 510 can also control camera 506 viawires 514 and/or filter wheel 508 via wires 516. Electronic controller510 can also control the heating of array 204 by supplying electriccurrent via wires 518 a, 518 b.

The LEDs 502 a, 502 b can be turned on or off (e.g. strobed) orintensity modulated and gated relative to the image capture/recording bythe electronic camera 506. The oblique illumination described in thisembodiment is advantageous because it minimizes direct illumination ofthe camera 506 by light from the LEDs 502 a, 502 b and only thescattered or emitted light from the array through-holes 504 is receivedby the camera 506, resulting in a high contrast image because of thelower background light.

Alternatively, the through-hole array 202 is illuminated through athrough a dichroic filter and the fluorescence emission from thethrough-hole array 202 is reflected from the filter and directed towardsan optical detection element.

Images of the through-hole array 202 are captured by, the camera 506 andprocessed to extract information on biological or chemical activity ineach through-hole 504 of the through-hole array 202. For example,fluorescent images of the PCR reaction at the primer annealingtemperature taken at each temperature cycle can be processed withwell-known image processing algorithms for quantitative assessment oftranscript copy number in each channel of the array. Such algorithms aredescribed in publications such as U.S. Pat. Nos. 6,814,934; 7;188,030;7,228,237; and 7,272,506.

Alternatively, epi-illumination schemes well-known in the art could beimplemented to achieve a performance similar to the transilluminationscheme thus described. An exemplary embodiment is described in FIG. 6wherein the dual LED light source 502 a, 502 b of FIG. 5 is replaced bya single, light source: 602 illuminating a through-hole array 202through an optical fiber bundle 604 and a pair of concave minors 606 a606 b for oblique illumination of through-hole array 202. One example ofthe light source. 602 is a tungsten arc lamp collimated and conditionedby neutral density or spectrally selective filters 608 prior toillumination of the through-hole array 202. As in FIG. 5, electroniccontroller 610 can control light source 602, camera 606, and filterwheel 608, as well as the heating of array 202 via wires 612, 614, 616,618 a, and 618 b.

In another embodiment, the dual LED light source 502 a, 502 b of FIG. 5is replaced by a multi-element LED array. The LED wavelength is chosento excite the fluorescent probe in the samples (such as the SYBR® Greendye, available commercially from sources such as Applied Biosystems ofFoster City, Calif. and Qiagen Inc. of Valencia, Calif.) and the LEDelement spacing is chosen to match the groupings of through holes. Forexample, in one embodiment of the through-hole array, subarrays 8×8through-holes are set on a 4.5 mm apart such that the total 3,072 holearray is comprised of 4×12 subarrays. In this embodiment an 8×12 arrayof LEDs would illuminate the through-hole array.

In yet another embodiment, the CCD-based imaging system is replaced witha fast scanning system. In one example of a fast scanning systemdepicted in FIG. 7A, a focused light beam 702 is scanned in twodimensions across a through-hole array 704 and the fluorescence emission706 from the array 704 is directed towards an optical detector 708 a.Fluorescence 706 from each through-hole 710 a is related to the lightbeam position on the array surface 712. In one embodiment, light source714 can move relative to the array 704 such that light beam 702 issubstantially perpendicular with array surface 712. Alternatively, lightsource 714 can be stationary while the angle of the light 712 ismodulated to focus on particular through-holes 710,

An alternative approach depicted in FIG. 7.13 involves scanning aline-focused light beam 702 b in one dimension across the array 704 anddirecting fluorescence emissions 706 a-706 g towards a two-dimensionaloptical (lector 708 b such as a CCD camera. Fluorescence from eachthrough-hole 710 is determined by instantaneous position of the focusedlight beam 702 on the platen surface 712.

High-Speed Through-Hole Array Cases

Another embodiment of a thermal control device 800 is depicted in FIGS.8A and 8B. The top and bottom surfaces (804 and 806, respectively) of acase 802 enclosing a through-hole array 202 are fabricated from thinrigid plates separated by a polymer spacer with a lengthwise slot forholding the through-hole array 202 along its edge to maintain a thinuniform gap between the top and bottom plates defining the case 802. Insome embodiments, at least one of the plates 804, 806 is lapped orground flat so as to provide intimate thermal contact with the flatblock 810 of a thermal cycler. Additionally or alternatively, aflexible, thermally conductive polymer 812 can be attached to the plateto provide the requisite intimate thermal contact with the thermalcycler block. In some embodiments, the top plate 804 is that it isoptically transparent for illumination of the through-hole and detectionof the resulting fluorescence emission from each microchannel of thearray 202.

A thin adhesive layer mechanically connects the two plates 804, 806 tothe spacing 808, forming a hermetic seal. To prepare the cassette forthermal cycling, the case is partially filled with an immiscible (eFLUORINERT® coolant), a through-hole array 802 loaded with PCR reagentsand nucleic acid sample is inserted into the case 802, and the case ishermetically sealed with a plug of UV curable epoxy 814 such as DYMAX®OP-29V. The assembled case or cassette is placed onto a flat blockthermal cycler 810 and thermally cycled according to a prescribedprotocol suitable for implementing the PCR method.

One difference between the presently discussed embodiment and previouscases is the use of a high thermal conductivity, low specific heatmaterial such as a metal, ceramic or diamond for platen instead of a lowthermal conductivity, high specific heat material such as glass. Therapid conduction of heat through the platen will decrease the thermalcycle time accordingly. If the both sides of the array package requireoptical transparency, then a material such as diamond is a reasonablechoice. A second option to reduce the thickness of the platen plates tominimize the temperature difference across the plate and therefore thethermal transfer impedance.

Through-hole Array Temperature Modulation by Electrically ConductiveCases

Another embodiment, depicted in FIG. 9, modifies the system 800 of FIG.8 to replace the flat block thermal cycler 810 with aconstant-temperature heat sink 910. The case 902 is fabricated with abottom platen 906 that is thermally and electrically conductive andplaced in intimate thermal contact with a constant temperature heat sink910 kept at a temperature substantially below the PCR annealingtemperature. For example, the heat sink 910 can be cooled to betweenabout −20° C. to about 30° C., which is below the PCR annealingtemperature range of about 35° C. to about 65° C. and above typicalcoolant pour temperatures of about −25° C. The bottom platen 906 iselectrically isolated from the heat sink 910 by a thin thermallyconductive, electrically insulating dielectric layer 912. Suitabledielectrics include glass, ceramics, and diamond.

The platen is electrically connected to an external current source and aelectronically controlled switch (not depicted) by electrical contacts(e.g. wires 916 a 916 b). Closing the switch causes electrical currentto pass through the platen, causing the platen to heat from ohmic lossin the metal. The thermal power P produced by this method is P=i² R.where i is the instantaneous current through the metal and R is theplate's bulk resistance.

Heat from the platen 906 flows partially into the heat sink 910 andpartially into the through-hole array 202 by way of the interveningfluid. Opening the switch stops the flow of current and production ofheat within the platen 910. Heat now flows out of the through-hole array202 and into the lower temperature heat sink 910. The rate of heattransfer into and out of the case 902 is governed by the thermalconductivity and specific heat of the materials between the array 202and the heat sink 910, as well as the thermal properties of the array202 and the heat sink 910 themselves.

To reach and maintain a specified array temperature, at least onethermal sensor in intimate thermal contact with the though-hole array202 can provide a temperature signal for controlling the duration andfrequency of the switch opening and closing, thereby modulating theelectrical current through the platen 906.

Various schemes are possible for feedback control of the current throughthe platen 906 based on modulation of the frequency and time duration ofthe opening and closing of the switch. One scheme depicted in FIG. 10Apulse modulation as it enables simplified and precise control of thearray temperature by changing the frequency of current pulses throughthe platen 906. If there is a large difference between the array 202 andset point temperature, the pulse frequency is high, driving more currenton average through the platen. As the temperature difference decreases,so does the pulse frequency, ultimately reaching zero when the set pointtemperature is reached. This control scheme is amendable to low powerswitching power supply electronics. An alternative scheme depicted inFIG. 10B is to have a fixed pulse frequency but vary the time durationof the current pulses in proportion with the difference between thethermal sensor and set point temperatures—the larger the temperaturedifference, the longer the current pulse duration.

FIG. 11A provides a one-dimensional thermal model for estimating thetemperature decrease of the platen 202 during joule heating by a currentpulse. It is assumed that the platen is heated instantaneously (relativeto the heat loss) when current is injected into the platen. Thethermophysical parameters of an exemplary soda lime glass ease 1104 a,1104 b and FLUORINERT® FC-77 coolant 1106 are depicted below:

Parameter FC-77 Glass Thermal conductivity (k)$0.057\frac{W}{m \cdot K}$ $1.05\frac{W}{m \cdot K}$ Specific heat (c)$1172\frac{J}{{kg} \cdot K}$ $840\frac{J}{{kg} \cdot K}$ Density (ρ)$1780\frac{kg}{m^{3}}$ $2500\frac{kg}{m^{3}}$It is assumed that the specific heat of the platen material (e.g. 317stainless steel) is substantially lower (e.g. by over an order ofmagnitude) While the thermal conductivity of the platent material issubstantially higher (e.g. by over an order of magnitude) and that theseparameters do not significantly impact the rate of heat loss from theplaten,

FIG. 11A depicts a cross section of an exemplary case 1102 enclosing aplaten 202 between two layers of soda lime glass 1104 a, 1104 b andFUJORINERT® FC-77 (1106). The ease is immersed in a liquid heat sink1108 as described herein held at a constant temperature (T_(sink)).

The following thermal model is derived based on the above assumptionsand describes the time dependence of the temperature difference betweenthe platen 202 and the heat sink 1108 when a pulse of current (i) heatsthe platen 202 by joule heating:

$T = {T_{sink} + {\frac{i^{2}{R \cdot \Delta}\; t}{m \cdot c}e^{\frac{{- 2}k}{\rho \cdot c \cdot L^{2}}t}}}$

In the above model, T_(sink) is the temperature of the heat sink, i isthe input current, R is the platen resistance, Δt is the pulse duration,m is the mass of the EV-77 fluid and the glass, c is the specific heatof the FC-77 fluid and the glass, k is the thermal conductivity of theFC-77 fluid and the glass, p the density of the FC-77 fluid and theglass, and L is the thickness of the FC-77 fluid and the glass.

Assuming a current pulse amplitude of 200 A. In one second pulsesthrough a platen of 10 mΩ resistance and combined FC-77 fluid and glassslide thickness of one millimeter, the platen 202 will reach an initialdouble-stranded DNA target melting temperature (FIG. 6d ) of 98.26° C.and decrease to an annealing temperature of 55° C. In ten seconds. Theramp up back to the melting temperature assumed to take less than onesecond. Based on these assumptions, a 40-cycle PCR protocol will takeapproximately 440 seconds or 7.3 minutes—substantially faster thanconventional flat block thermal cycler where heat is conducted throughthe material comprising the walls of the liquid container to heat theliquid and back out again to cool the liquid. The approach proposed hereis fundamentally different in that the container itself is directlyheated to then heat the enclosed liquid by thermal conduction. Heat isremoved from the reaction volume by conduction through the platen 202and encapsulating liquid 1106 and glass slides 1104 a, 1104 b to theexternal heat sink 1108.

In another example, again assume that the plate has a resistance of 10mΩ and that the dominant thermal loss is through the PC-77 liquid. Acurrent pulse of 500 A and 5 V applied to the plate for 150 ms will heatthe plate to 95.87° C. The average power of into the plate is 375 W (5V×500 A×0.15 s), which can he provide by a standard pulsed power supply(e,g. a power supply from standard photgraphic strobe flash devices).

Compensation for Spatially Non-Uniform Heating

Injection and extraction of thermal energy to ensure spatially uniformheating and cooling of the fluidic array is important for uniform PCRamplification. Typically, if the cassette is heated and cooled on a flatthermal cycler block, the higher thermal conductivity of the polymerspacer relative to the FLUORINERT® liquid and the intimate thermalcontact between the case spacer and the metal array relative to theFLUORINERT® liquid in contact with the: array causes the temperature atthe edges of the array to lead the temperature change at the center ofthe array. This temperature gradient across the array can be as large as1-2° C. while the package is heated or cooled.

One approach to minimizing this thermal gradient is to differentiallyheat/cool the thermal package such that the heat flux is greater in thecenter of the array compared with the edges. This strategy is readilyaccommodated in the present embodiment by electrically segmenting theconductive plate such that there is a slightly greater electricalcurrent through the central region of the plate than along its edges. Inturn, the plate temperature mirrors the spatial distribution ofelectrical current through the plate.

An exemplary case 1200 is depicted in FIG. 12. The conductive portion ofcase 1200 includes three conductive strips 1202 a, 1202 b, 1202 cseparated by two regions 1204 a, 1204 b of electrically and thermallyinsulating material. The insulating material can be an adhesive, air, oran insulating liquid, depending on the positioning of the conductivestrips within the case 1200. A different current through each strip 1202a, 1202 b, 1202 c is achieved by allowing each strip 1202 a, 1202 b,1202 c to have a different intrinsic resistance than its neighbors or bychanging the resistance with external resistors between the strips 1202a, 1202 b, 1202 c and current supply.

For example, if external resistors are used, an infrared sensitivecamera can be used to image the array 202 with equal value resistors toestablish a temperature baseline as the plate is heated and cooled. Theresistor values can then be changed to minimize the observed temperaturegradient across the array 202 as the temperature changes. Because thepower dissipated is proportional to electrical resistance, and theobserved temperature gradient is proportional to the spatialdistribution of dissipated power, small changes in the external resistorcan decrease the temperature difference across the plate.

Direct Heating of the Through-Hole Array

In another embodiment, the through-hole array is directly heated bypassing an electrical current through the array substrate itself. Asshown in FIG. 3, case 200 can be modified by inclusion of an electrode312 such that the array 202 makes electrical contact with the electrode312 on insertion into the case 200. The plug of UV curable epoxy can bereplaced with a mechanical plug 314 providing an electrical contact tothe array 202. The case 200 can be held vertically to prevent the liquidfrom leaking from the case. The electrodes 312, 314 embedded in the case200 package provide the connection to the external current supply 304 a,304 b and electrical switch 306. A temperature sensor in intimatethermal contact with the array 202 can provide temperature informationfor feedback control of injection of the electrical current into thearray 202. Control schemes such as those described herein can be appliedto control the array temperature. Because of the potential forelectrical interference with the thermal sensor, the sensor can, in someembodiments, be read-out only between current pulses applied to thearray 202.

The case 200 can be placed on a constant temperature heat sink at atemperature substantially less than the PCR annealing temperature tocool the array after heating. The rate of cooling is directlyproportional to the difference in temperature between the block and thelowest temperature of the PCR process. Since the annealing temperatureis typically between 50-60° C., a heat sink of lower temperature istypical.

Heating of Through-Hole Arrays by Radio Frequency (RF)

In another embodiment of the invention, the through-bole array is heatedvia radio waves (e.g., microwaves). In such an embodiment, the case 200can be fabricated from dielectric, non-conductive materials, while thethrough-hole array 202 is formed from conductive materials. Suitable RFheating frequencies include frequencies between about 0.1 MHz and about10 MHz.

The heating of through-hole arrays can be controlled according to thecontrol schemes described herein. The cooling of through-hole arrays canbe effected by a variety heat sinks as described herein.

Heating of Through-Hole Arrays by Infrared Waves

In another embodiment, the samples inside the through-holes are directlyand/or selectively heated using near-IR radiation. Water is a strongabsorber of near-radiation around 1050 nm, whereas FC-77 liquid andother perfluorinated hydrorcarbon oils are highly transmissive in thenear-infrared region. The near-IR radiation is provided by an infraredLED array or other narrowband IR radiation source located on theopposite side of the array from the CCD camera. Cooling is provided bycirculating chilled perfluorinated hydrorcarbon oils around the sample.

Thermal Cycling of Through-Hole Array Through Direct Contact withLiquids

In another embodiment, the heating and cooling of the samples in athrough-hole array is caused by alternating the flow of hot and coldimmiscible liquid into a chamber containing the OPENARRAY™ device. Insome embodiment, the chamber is a variant of the through-hole arraycases described herein.

FIG. 13 depicts an exemplary embodiment of a system 1300 for thermalcycling a through-hole array 1302. The system includes a case 1304 forreceiving the through-hole array 1302. The case 1304 includes a firstport 1306 a located at a first end of the case 1304 and a second port1306 b located at a second end of the case 1304. Ports 1306 a 1306 b areconnected to a fluidic circuit including a pump 1308, a hot tank 1310,and a cold tank 1312. Valves 1314 a, 1314 b are selectively actuated toallow for the alternative flow of hot fluid and cold fluid through thecase 1304.

The case 1304 can include a plurality of veins 1316 a-f to promote amore uniform flow rate over the through-hole array 1302. In someembodiments, the samples are protected by first covering thethrough-hole array 1302 in a thin layer of an inert oil that isimmiscible to the liquid used to heat and cool the through-hole array1302.

Heat Sinks

A variety of heat sinks can be used to cool the through-hole arrays andcases provided herein. For example, the heat sink can be a cooled blockcomposed of a thermally-conductive material such as a metal. The blockcan be passively cooled or actively cooled by flowing air or liquidthrough channels or veins located within or on a surface of the block.Additionally or alternatively, the block can be or can be coupled with aPeltier cooling element.

The heat sink can additionally or alternatively be a fluid bathcontaining, for example, a liquid or an ice slurry. The fluid bath canbe a stationary bath or can be circulated, e.g. through a refrigeratorpump.

The heat sink can also be a gas stream directed over case 200. Forexample, one or more fans or jets can be arranged to flow a cool streamof a gas over the case. The gas can be, e.g., air or liquid nitrogen.

The case can be in continuous contact with the heat sink or contact canbe variable. For example, the case 200 can be completely removed fromthe heat sink. (e.g., by lifting the case out of a fluid bath) when thearray is heated. Alternatively, the fluid bath or gas stream can becirculated when the case 200 is cooled and not circulated when the case200 is heated.

Methods of Thermal Cycling

As described herein, the devices herein can be used for a variety ofthermal cycling methods. An exemplary embodiment is depicted in FIG. 14.

In step S1402, a microfluidic array provided. The microfluidic array canin some embodiments be a though-hole array as described herein. However,other microfluidic devices can be used including glass, plastic, metal,or silicon plates containing a plurality of micro wells etched in asurface, but no extending to an opposing surface.

In step S1404, the microfluidic array is loaded. The microfluidic arraycan be loaded by a variety of known methods including dip loading,droplet dragging, and the use of one or more pipettes. Variousmicrofluidic array loading techniques are described in U.S. Pat. Nos.6,306,578; 6,387,331; 6,436,632; 6,716,629; 6,743,633; 6,893,877;7,332,271; and U.S. Patent Application Publication Nos. 2001/0055765;2002/0151040; 2002/0192716; 2003/0124716: 2003/0180804; 2004/0037748;2004/0171166; 2004/0191924; 2004/0208792; 2005/0059074; 2005/0079105;2005/0148066; 2005/0230213; 2006/0183171; 2007/0003448; and2008/0108112. Suitable devices for loading microfluidic arrays includethe OPENARRAY® AUTOLOADER™ device available from BioTrove, Inc. ofWoburn, Mass.

In steps S1406, the microfluidic array is placed in an appropriate case.In step S1408, the case is filled with an immiscible fluid (e.g.FLUORINERT® FC-77 fluid). The case is then sealed in step S1410. Forexample, the case can be sealed inserting a mechanical plug or applyingan adhesive to fill an opening in the case or bend one or more walls ofthe case. The adhesive can be a UV-curable adhesive as discussed hereinand in U.S. Patent Application Publication No. 2004/0208792.

In step S1412, the case is placed in a thermal cycler. The case can bepositioned to mate with a particular thermal cycler geometry. Forexample, one or more electrical contacts on an exterior surface of thecase can be aligned with corresponding contacts in the thermal cycler.Alternatively, one or more ports on a case can be coupled with outletsfor a fluidic circuit in the thermal cycler. The thermal cycler can, insome embodiments, include one or more clamps or locks to hold the caseagainst electrical contacts and/or the heat sink. Suitable clampsinclude one or more fingers described in U.S. Patent ApplicationPublication No. 2006/0094108. Such fingers advantageously apply pressureto the case without obstructing imaging of the array through atransparent wall of the case.

in step S1414, the microfluidic array is heated. This heating can beaccomplished by a variety of methods as described herein including;Joule heating of the case, Joule heating of the microfluidic array,infrared heating, radiation heating, and flowing heating fluid throughthe case. The microfluidic array is heated to a desired temperature(e.g. 98.26° C. the melting temperature for double-stranded DNA).

In step S1416, the micron array is cooled. This cooling can beaccomplished by a variety of method as described herein including:removing a heat source, exposing the case to ambient air, exposing thecase to Chilled liquid, placing the case in contact with a chilledsurface, flowing a chilled liquid through the case. The microfluidicarray is heated to a desired temperature (e.g. 55° C.—the annealingtemperature for double-stranded DNA).

In step S1418, the microfluidic army optionally imaged. The imaging canbe in accordance with real-time PCR method as described in U.S. Pat.Nos. 6,814,934: 7,188,030; 7,228,237; and 7,272,506. The heating,cooling, and imaging steps can be repeated.

EQUIVALENTS

The foregoing specification and the drawings forming part hereof areillustrative in nature and demonstrate certain preferred embodiments ofthe invention. It should be recognized and understood, however, that thedescription is not to be consulted as limiting of the invention becausemany changes, modifications and variations may be made therein by thoseof skill in the art without departing from the essential scope, spiritor intention of the invention. Also, various combinations of elements,steps, features, and/or aspects of the described embodiments arepossible and contemplated even if such combinations are not expresslyidentified herein.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications, andother references cited herein are hereby expressly incorporated hereinin their entireties by reference.

1.-47. (canceled)
 48. A through-hole array comprising: a platen having afirst end region a second end region; a plurality of strips spanningfrom the first end region to the second end region; and a plurality ofthrough-holes located on one or more of the plurality of strips.
 49. Thethrough-hole array of claim 48, wherein the strips are substantiallyparallel.
 50. The through-hole array of claim 48, further comprising:one or more slots, each slot separating two of the plurality of strips.51. The through-hole array of claim 48, wherein the platen is formedfrom a conductive material.
 52. The through-hole array of claim 51,wherein the conductive material selected from the group consisting of:copper, gold, silver, nickel, iron, titanium, steel, and stainlesssteel.
 53. The through-hole array of claim 48, wherein the plurality ofthrough-holes located on one of the plurality of strips are arranged ina single column.
 54. The through-hole array of claim 48, wherein thethrough-holes have a hydrophilic interior.
 55. The through-hole array ofclaim 48, further comprising: two outer layers of hydrophobic materialcoupled to a top and a bottom surface of the plurality of the strips.56. The through-hole array of claim 48, wherein each of the plurality ofthrough-holes has a volume less than 100 nanoliters.
 57. A container forthermal cycling a plurality of samples in a microfluidic array having aplurality of through-holes arranged on a plurality of strips, thecontainer comprising: a plurality of walls defining an interior volume;and a plurality of fingers configured to contact the strips when themicrofluidic array is inserted in the container.
 58. The container ofclaim 57, further comprising: a pair of electrically-conductive contactslocated on an exterior surface of the container, the contacts incommunication with the fingers.
 59. The container of claim 57, whereinthe fingers are configured to contact the microfluidic array at a firstand a second end of each of the plurality of strips.
 60. The containerof claim 57, wherein at least one of the plurality of walls is opticallytransparent.
 61. The container of claim 57, wherein the plurality offingers are comprised of a metal.
 62. A method for thermal cycling aplurality of samples, the method comprising: providing a through-holearray comprising: a platen having a first end region and a second endregion; a plurality of strips spanning from the first end region to thesecond end region; and a plurality of through-holes located on one ormore of the plurality of strips; loading the plurality of samples intothe plurality of through-holes; placing the though-hole array in acontainer, the container including a plurality of fingers configured tocontact the strips; applying a flow of electrical current across theplurality of strips; and terminating the flow of electrical current toallow the samples to cool.
 63. The method of claim 62, furthercomprising: placing the container in contact with a heat sink.
 64. Themethod of claim 63, wherein the heat sink is a fluid bath.
 65. Themethod of claim 64, wherein the fluid bath is chilled.
 66. The method ofclaim 63, wherein the heat sink is a Peltier element.
 67. The method ofclaim 62, further comprising: imaging the through-hole array. 68.-86.(canceled)